MATERIALS AND METHODS

Crude oil: The Crude oil used was gotten from the Directorate of Petroleum Resources Port-Harcourt, Rivers State Nigeria.

Soil: The soil samples that was used in this study were obtained from the Agric Farm, Department of Agriculture, University of Nigeria, Near Green House.

Microorganism: Two microorganisms were used in the course of the research. The Pseudomonas species was gotten from the culture collection Center Department of Microbiology, University of Nigeria, Nsukka while the Azotobacter vinelandii was isolated from the soil around the postgraduate laboratory, Department of Biochemistry, University of Nigeria, Nsukka using Azotobacter vinelandii specific media.

Preparation of media
Preparation of Broth for the isolation Azotobacter vinelandii using specific media: The media for the isolation of Azotobacter vinelandii media was prepared as follows; Sodium benzoate (1.0 g), Di-Potassium hydrogen phosphate (K₂HPO₄) (0.5 g) and Mannitol (0.5 g) were put in a 1 L conical flask and small quantity of distilled water was added and the volume was brought to 999 mL the mixture was thoroughly mixed and autoclaved for 15 min at 15 psi pressure-121°C. Cool to 45-50°C after that, ethanol (1 mL) was aseptically added and then aseptically distributed into sterile tubes or flasks.

Preparation of Pseudomonas maintenance media: The Pseudomonas maintenance media was prepared as follows:

Preparation of trace element solution: The trace element was prepared by adding Boric acid (H₃BO₃) 0.232 g, Zinc sulphate heptahydrate (ZnSO₄·7H₂O) 0.174 g, Ferrous Ammonium sulphate (FeSO₄(NH₄)₂SO₄·6H₂O) 0.116 g, Cobalt (II) Sulphate heptahydrate (CoSO₄·7H₂O) 0.096 g, Ammonium molybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O) 0.022 g Copper sulphate pentahydrate (CuSO₄·5H₂O) 8.0 mg and MnSO₄·4H₂O 8.0 mg were added to a 1 L conical flask and made up to make with distilled water.

Determination of the remaining Total Petroleum Hydrocarbon (TPH): One gram of soil was put in a test-tube and ten milliliter (10 mL) of Chloroform/Ethanol mixture (1:1) was added. The mixture was agitated for 5 min and then allowed to stand for 10 min. The sample was then filtered and the absorbance of the filtrate was taken at 520 nm using chloroform/Ethanol mixture (1:1) as a blank. The quantity of crude oil was estimated using a crude oil standard curve.

Determination of degraded TPH will be done by calculation: The amount of crude oil degraded was estimated by simple calculation. This was done by subtracting the initial concentration of crude oil from the final concentration crude oil.

Experimental design: The experiment was setup in 6 groups each group contain various concentrations of crude oil (0.1, 0.5, 1.0, 5.0 and 10%). Each group contain either Pseudomonas sp or Azotobacter sp. with amendment. The groups are as follows:

Two hundred grams of soil was put in a conical flask and groups 4-5 was impregnated with 10 mL of 0.1 M of their salt solution, the soil was mixed very well to ensure proper spreading of the salts. It was then autoclaved to kill the organisms present in the soil and allowed to cool to room temperature. After that 1 mL of the microbial broth containing Pseudomonas sp. Except for the group 3 that contain 0.5 mL of each organism. After stirring, the experiment was monitored for two weeks.

Statistical analysis: The results of the experiments were analyzes statistically and are presented as Mean±SEM, the linear regression model was used to analyze the rate of breakdown, while the non-linear regression model was used to determine the V_max and the K_m the level of significant was determined at p<0.05. The graphpad prism v 6 was used for the analysis.

RESULTS

The result in Fig. 1-6 show the graphs for the breakdown of crude oil using different nitrogen sources. All the graphs show decreases in crude oil (TPH) as time increases in other words, it was observed that TPH decreased with time. The curves in Fig. 2 were observed to tilt more to the horizontal signifying very little decrease as the time increases.

Fig. 1:	Degradation of various concentration of TPH using Pseudomonas sp. alone

Fig. 2:	Degradation of various concentration of TPH using Azotobacter vinelandii alone

The linear regression was used to determine the slope of the graphs, which represent the rate of degradation of crude oil will the R-square value was used to identify how accurate (perfect) the curve is the data was presented in Table 1.

The result in Table 1 shows the rate of degradation crude oil as obtained from Fig. 1-6. The equation of the line of best fit was determined and the slope which is the rate of degradation. The R-square values were also seen to be more than 0.7 or one could say that the curves were more than 70% perfect.

Fig. 3:	Degradation of various concentration of TPH using a consortium of Pseudomonas sp and Azotobacter vinelandii

The negative values of the slope simple shows the rate of reduction. It was also observed that the value of the slope increases with increase in the percentage of crude oil with except in the group containing Azotobacter vinelandii which fluctuate as the percentage of crude oil increases. Also the group containing Nitrite and nitrate also showed a decrease in the slope after 5.0% crude oil contamination.

Fig. 4:	Degradation of various concentration of TPH using Pseudomonas sp and Ammonium salt

Table 1:	Rate of degradation of crude oil, which was obtained from Fig. 1-6

Table 2:	Vmax and Km of Pseudomonas with various nitrogen sources

Fig. 5:	Degradation of various concentration of TPH using Pseudomonas sp and Nitrite salt

Fig. 6:	Degradation of various concentration of TPH using Pseudomonas sp and Nitrate salt

To determine the Vmax and Km, the graph of rate of degradation (slope) against substrate concentration were plotted and represented in Fig. 7, 9, 11, 13, 15 and 17. Figures were observed to be sigmoid in shape according to the Michealis -Mentens pattern, Fig. 9 falling out of place. due to the fact that the points in the graph could not converge. In order to linearize the curves, a plot of the inverse of the rate of degradation against the inverse of the substrate concentration was done and represented in Fig. 8, 10, 12, 14, 16 and 18. The values of V_max were obtained as the inverse of the value of the rate when substrate concentration is zero, while the K_m were obtained as the negative of the inverse of the value of substrate when the rate is zero.

Fig. 7:	Graph of the rate of breakdown of TPH using Pseudomonas sp. alone against substrate concentration

Fig. 8:	Graph of the inverse of the rate of breakdown of TPH using Pseudomonas sp alone against the inverse of the substrate concentration

The values of V_max and K_m were represented in Table 2.

The Table 2 shows the result of the V_max and K^m of Pseudonmonas sp. and the various amendment. The result reveals that the introduction of nitrogen sources reduced both the V_max and K_m. Azotobacter vinelandii’s V_max and K_m were not determined because the point were not converging and the line of best fit gave an R-square value of 0.039, which means that the curve is about 3.9% perfect. The consortium of Pseudomonas sp. and Azotobacter vinelandii showed lowest V_max value (0.090±0.009) and highest K_m value (0.584±0.239) in the groups containing nitrogen sources. The V_max: K_m ratio showed that Ps+NO₂>Ps+NH₄>Ps+NO₃>Ps>Ps+Az.

Fig. 9:	Graph of the rate of breakdown of TPH using Azotobacter vinelandii alone against substrate concentration

Fig. 10:	Graph of the inverse of the rate of breakdown of TPH using Azotobacter vinelandii alone against the inverse of the substrate concentration

Fig. 11:	Graph of the rate of breakdown of TPH using a consortium of Azotobacter vinelandii and Pseudomonas sp against substrate concentration

Fig. 12:	Graph of the inverse of the rate of breakdown of TPH using Azotobacter vinelandii and Pseudomonas sp. against the inverse of the substrate concentration

Fig. 13:	Graph of the rate of breakdown of TPH using Pseudomonas sp. and nitrate salt against substrate concentration

Fig. 14:	Graph of the inverse of the rate of breakdown of TPH using Pseudomonas sp. and nitrate salt against the inverse of the substrate concentration

Fig. 15:	Graph of the rate of breakdown of TPH using Pseudomonas sp. and nitrite salt against substrate concentration

Fig. 16:	Graph of the inverse of the rate of breakdown of TPH using Pseudomonas sp and nitrite salt against the inverse of the substrate concentration

DISCUSSION

Pseudomonas sp. has been employed in the bioremediation of crude oil polluted environments (Onwurah et al., 2007). One of the challenges facing microorganisms during bioremediation is the availability of macro-nutrients, such as; nitrogen and phosphorus (Bahadur et al., 2014) Pseudomonas sp has the ability to solubilize phosphorus in the soil (Delgado et al., 2014) but its major challenge is the availability of nitrogen. Pseudomonas sp. has also the ability to depend on crude oil for energy and biomass production (Goteti et al., 2014). When, nitrogen is not available in the soil, the organism begins to decrease in population, because of the inability to synthesize nucleotides and amino acids of which nitrogen is a major component.

Fig. 17:	Graph of the rate of breakdown of TPH using Pseudomonas sp. and ammonium salt against substrate concentration

Fig. 18:	Graph of the inverse of the rate of breakdown of TPH using Pseudomonas sp. and ammonium salt against the inverse of the substrate concentration

Therefore to sustain the survival of the Pseudomonas in polluted sites, nitrogen sources need to be introduced, since the pollutants (crude oil) acts as a carbon source (Palanisamy et al., 2014). This present study sets out to find the effect of different nitrogen sources on the biodegradation ability of Pseudomonas sp. Different nitrogen sources both organic and inorganic was used for this purpose. The graphs of the concentration of crude oil against time which was used to determine the rate of degradation showed that Azotobacter vinelandii is not a good degrader of crude oil when used alone. In Fig. 2, the curves in the graph were tending towards the horizontal, when compared with the others. It was generally observed that the rate of breakdown of crude oil increases with increase in the concentration of crude oil, with exception of Azotobacter vinelandii alone, which did not show any pattern. Also the presence of nitrate and nitrite showed a decrease in the rate of degradation from 5.0% crude oil contamination.

On determining the V_max and K_m of the individual groups, the group containing Azotobacter could not converge so it was practically impossible to determine the V_max and the K_m for the organism alone. In addition, the line of best fit in figure 10 had an R² value of 0.039, which could be interpreted as 3.9% accurate. This also support the claim that Azotobacter even though it breaks down the crude oil to some extent, is not an effective degrader.

The results showed that the introduction of nitrogen sources cause a reduction in V_max and K_m of the organisms in all the groups. This showed that to some extent, the introduction of nitrogen causes some level of inhibition although, it does not terminate the organisms. The decrease in K_m, when nitrogen source was introduced showed that nitrogen on the other hand increases the affinity of the organism for the substrate (crude oil).

From the result, Pseudomonas with nitrite showed the lowerest V_max value (0.106±0.011) and K_m value (0.151±0.089). Since, the decrease affects both V_max and K_m by different degrees, it becomes necessary to use another parameter that will cushion the effect and give a better comparable parameter. In this regard, the V_max: K_m ratio was determined. This ratio shows the level of dilution per quantity of substrate that will produce maximum or optimum activity. In other words, the lower values of this ratio showed that optimum activity could be obtained even at higher concentration of the substrate (crude oil) and this means that the organism with low V_max: K_m ratio can also do better at high concentration of crude oil. In this experiment, the order of the ratio is Ps+Az>Ps>Ps+NO₃>Ps+NH₄>Ps+NO₂, making the consortium of Pseudomonas sp and Azotobacter vinelandii a better option rather than the use of in organism sources of nitrogen.

CONCLUSION

The argumentation of the breakdown of crude oil by Pseudomonas with an organic nitrogen source, such as; Azotobacter vinelandii was shown to produce better results. In addition, the sustainability of the consortium and its environmentally friendliness have made the consortium a better choice over the inorganic nitrogen sources that have the ability to increase cost and introduce other pollutant into the environment.